RE: [Histonet] Wax removal incomplete

From:Luis Chiriboga

Likewise,  I have also wondered about these "plasticizers" added to
paraffin. There really is no literature (that I can find) discussing this
issue. We recently were studying Pten expression in skin samples dating as
far back as the late 70's. Interestingly, samples collected in the mid
1980's did not deparaffinize as well as more recent (and older) samples.  It
turned out that these samples required substantial heating (upwards of 80C,
normally we do at 60C) to generate IHC labeling.
In regards to the Raman paper, I have done quite a bit of similar work with
FTIR in FFPE tissue and would like to point out some critical points.  Both
Raman and FTIR  techniques are very sensitive to molecular structure.
Unfortunately the authors do not describe or show from where
(histologically) they collected their spectra. This can influence the
resulting spectra quite dramatically (remember they are using a 2um spot
size).  Although both techniques are considered "fingerprinting" methods in
that they can identify compounds based on their unique vibrational
signatures, they also suffer from the averaging effect. Thus, other
compounds (many compounds will have C-C stretching and CH2 bending and
deformation modes) that produce similar spectral signatures will be averaged
into the overall spectrum.


Luis Chiriboga Ph.D.
NYU Cancer Institute and
Bellevue Hospital Center
New York University School Of Medicine
Department Of Pathology 4W27
462 First Avenue
New York, N.Y. 10016
W(212) 562-4667.
F(212) 263-2041

-----Original Message-----
[]On Behalf Of LEWIS,
Sent: Tuesday, February 15, 2005 9:09 AM
To: John Kiernan; Histonet
Subject: RE: [Histonet] Wax removal incomplete

I often wondered about this due to the "plastic "polymers added to the
different types of paraffins. Perhaps it's these "polymers" that are not
being removed with the typical solvents.

Interesting ...

Thanks John !

Mark A. Lewis B.A., H.T.(ASCP)
Product Specialist
Anatomical Pathology
Clinical Diagnostics
Thermo Electron Corporation
171 Industry Drive
Pittsburgh, Pa. 15275
Phone:  412-747-4013
Fax       412-788-6557

-----Original Message-----
[] On Behalf Of John Kiernan
Sent: Monday, February 14, 2005 1:40 PM
To: Histonet
Subject: [Histonet] Wax removal incomplete

A recent publication from Ireland shows (by laser confocal
Raman microspectroscopy, no less) that wax removal from
sections, as usually practised, is always incomplete.
The authors were able to remove all the paraffin, however,
with hexane. The other things they tried were xylene,
"histoclear" followed by hot antigen retrieval, and
"trilogy" (a product that combines dewaxing and retrieval
- ? a solvent-detergent mixture). All three failed to
remove all the wax, even with repeated, prolonged
For hexane, 18 hours were needed for complete was removal.

The investigators found that the residual paraffin
seriously interfered with the Raman spectra of
rehydrated sections. They are investigating the Raman
technique (which they explain fairly simply) for early
diagnosis of tumours. They also claim stronger
immunostaining after dewaxing in hexane (18h) than after
xylene (18h). They tested only one antibody (to
and the pair of photos doesn't make the point very

They authors also state that hexane is less toxic than
xylene. I can add that the least expensive n-hexane is
a little cheaper than xylenes (mixed isomers) in one
catalogue checked.  Both solvents are flammable, and
n-hexane (BP 69C) is more volatile than xylene. Both
solvents are miscible with 100% alcohol.

The reference is:
O'Faolain E et 6 al. (2005) Raman spectroscopic evaluation
of efficacy of current paraffin wax section dewaxing
agents. J. Histochem. Cytochem. 53(1): 121-129.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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